Project Summary/Abstract Timely and accurate regulation of gene expression is required for proper growth, development, and response to environmental stimuli. A complete understanding of the mechanisms employed to regulate gene expression is necessary to combat the aberrant regulation that underlies many human developmental conditions and ailments, including cancer. The long-term goals of this proposal are to determine the mechanics of RNA synthesis by DNA-dependent multi-subunit RNA polymerases (RNAPs), the regulation imposed on RNAP by conserved protein factors, select template sequences and chromatin-barriers, and to specifically characterize the available mechanisms to halt RNA synthesis and terminate transcription. The proposed experiments take advantage of an established in vivo and in vitro archaeal transcription system from the marine hyperthermophilic organism Thermococcus kodakarensis. Archaea offer the advantages of far less complexity but homology in many features of human molecular biology, specifically conservation of RNAP structure and function and histone-based chromatin. We will characterize factor-dependent termination, polarity, the consequences of DNA damage on transcription elongation and termination, and the role of global transcription regulation in archaeal physiology. The results obtained will determine the structures and sequences that support transcription elongation complex stability, describe the mechanics of termination and allow comparisons of termination mechanisms in each domain of life, and provide insight to control and ultimately manipulate gene expression to counter a variety of diseases and ailments affecting human health.